Campylobacter jejuni is currently the most common bacterial cause of diarrheal disease in humans in the United States. It has also recently been strongly implicated as one of the most common infections to precede the development of Guillain-Barre syndrome. Thus far, only one C. jejuni toxin, cytolethal distending toxin (CDT), has been definitively proven to exist. Recent work has shown that the C. jejuni CDT is a member of a new family of toxins that can cause a G2 cell cycle block in certain mammalian cultured cells. However, little is known about the specific functions of this toxin and how it contributes to C. jejuni disease. The long-term goals of this proposal are to understand how CDT causes a cell cycle block and to relate this understanding to disease. In particular, the investigator plans to examine the functional roles of 3 CDT proteins (CdtA, CdtB, and CdtC) in causing the G2 block. The proposal seeks to elucidate the target(s) of CDT in sensitive cells, and determine the roles of the Cdt proteins in causing the toxic effect. The specific aims of the proposal are to test the hypothesis that one or more of the Cdt proteins function to bring about the G2 block in various types of eucaryotic cells. In Aim 1, a variety of specific mutations will be introduced into each of the Cdt proteins. The effects of the mutations on toxicity and on selected other properties will be tested. In Aim 2, yeast 2-hybrid techniques will be used to identify HeLa cell proteins that interact with Cdt proteins. In Aim 3, a yeast expression system will be exploited to identify how Cdt causes the G2 block. In Aim 4, a HeLa cell expression system will be used to complement and extend the studies in the first 3 aims. In summary, these studies are designed to increase our understanding of CDT and the mechanism by which it causes a G2 block.